Mammals achieve dosage compensation by transcriptional silencing of one X-chromosome in early female development. X-chromosome inactivation (XCI) is controlled by a master locus known as the "X-inactivation center," a cis-acting center which is both necessary and sufficient to direct X-chromosome counting, choice, and initiation of silencing. Our long term goal is to understand the molecular underpinnings of each step. It is currently known that Xist RNA, a non-coding product of the X-inactivation center, "paints" the inactive X and is required to initiate global silencing of that X. Work from the last funding period led to the discovery of two novel non-coding RNA loci at the X-inactivation center. Tsix, a gene antisense to Xist, inhibits Xist expression and designates the future active X. Xite, a locus associated with low-level intergenic transcripts, positively regulates Tsix expression. Thus, all known regulatory elements at the X-inactivation center make non-coding RNA. The proposed research will dissect the mechanism of action by Tsix and Xite. Specifically, the proposed research will (1) determine how Xite and Tsix inhibit the spread of Xist RNA, (2) define the molecular basis of X-inactivation imprinting and choice, and (3) test if Xite is the Xce ("X-controlling element"), the long-elusive modifier of XCI allele ratios which has been implicated in unfavorable skewing of XCI in female carriers of X-linked disease. As one of very few antisense loci in mammals, Tsix serves as a paradigm for antisense regulation. As one of only two known functional intergenic transcription elements in mammals, Xite will advance a limited knowledge of this emerging intergenic phenomenon. Information gained from the research program will also have significant impact on human genetics, cancer, and animal cloning, as well as on the basic biology of chromosomes, chromatin structure, and transcriptional regulation.